We are studying the reactions required for DNA replication in vitro using proteins encoded by T4 bacteriophage. With single-stranded phi X 174 DNA as a model template, we find that ribooligonucleotide primers are made by the T4 gene 41 protein and a second protein controlled by gene 61. These primers are elongated to form duplex circular molecules by the gene 43 DNA polymerase, gene 45 protein, and the complex of gene 44 and 62 proteins (a DNA-dependent ATPase). If the gene 32 DNA binding protein is also present, the DNA is further elongated by strand displacement synthesis yielding rolling circles with long tails. The sequence of the principal primer made in the presence of the four ribonucleoside triphosphates is pppACN3N4N5, with G and to a lesser extent C and/or U in positions 3, 4, and 5. RNA primers are initiated at many different positions on the template.